PROJECT SUMMARY: Cleft palate patients (1:1000 live births) all undergo palate repair; however, 60% de- velop complications including oronasal fistula (ONF) formation. ONF formation leads to poor feeding and speech. Cleft palate repair involves rotating palate mucosal flaps into the cleft and healing is affected by physi- cal trauma from the tongue and a bacteria laden environment. Currently, cleft palate repair surgeries lack a regenerative reconstructive option, require multiple re-repairs due to ONF and increase cost and morbidity. Surgeons use allograft dermal implants, despite the risk of HIV/prion transmission, to act solely as a barrier to reduce ONF. Our group has developed a novel murine phenocopy of ONF, and identified a unique immuno- modulatory approach to directing the ONF healing process in a pro-regenerative program. FTY720, an active biolipid, targets the sphingosine pathway to preferentially attract pro-regenerative monocytes and macrophag- es to improve ONF healing. Our long-term goal is to develop an immunoregenerative approach to improve pal- ate wound healing. The overall objective in this application is to determine the mechanism of ONF healing and use FTY720 nanofibers to tune the monocyte and macrophage migration in a pro-regenerative fashion. The central hypothesis is that inflammatory monocytes and macrophages are the predominant response during oral cavity wound healing and that delivery of FTY720 preferentially attracts pro-regenerative monocytes and mac- rophages leading to improved wound healing. FTY720 signals through the sphingosine pathway, increasing the recruitment of pro-regenerative monocytes and macrophages. The rationale for the proposed research is that a comprehensive and mechanistic understanding of the inflammatory response during ONF formation will provide therapeutic targets aimed at altering pro-regenerative monocyte migration. Guided by strong prelimi- nary data, including a paper demonstrating significant reduction in ONF following FTY720 nanofiber delivery, the hypothesis will be tested by pursuing two specific aims: 1) Determine the requirement, timing and mecha- nistic contributions of the inflammatory response during ONF formation; 2) Engineer a nanofiber scaffold to provide controlled delivery of FTY720 to improve oral cavity wound healing using an immunoregenerative ap- proach. In Aim 1 we will identify the critical inflammatory cell recruitment to healing ONF, determine the re- quirement of monocytes, and identify alteration in the cytokines and reparative gene pathways following ONF formation. In Aim 2, we will test the optimal FTY720 delivery strategy, the ability of FTY720 nanofiber to direct pro-regenerative ONF wound healing with and without monocytes, and determine the effects of FTY720 nano- fiber on the cytokines and reparative gene pathways. The proposed research is innovative by mechanistically describing monocyte migration in an ONF murine model that allows testing of immunomodulatory strategies. The proposed research is significant because it that FTY720, an FDA-approved drug approved to treat multiple sclerosis, could be fast tracked to clinical trials to reduce ONF formation.